PROJECT SUMMARY Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and related diseases in animals, mainly in sub-Saharan Africa. T. brucei undergoes life-cycle-specific differentiation between the insect vector (tsetse) and mammalian hosts. In the mammalian hosts, ?Bloodstream Form (BF)? trypanosomes proliferate extracellularly in the vascular system and the surfaces of trypanosome cells are coated with Variant Surface Glycoprotein (VSG). BF trypanosomes escape the host immune response through sequential expression of VSGs, a process known as ?antigenic variation?. Two mechanisms that underlie T. brucei antigenic variation, monoallelic VSG expression and periodic VSG switching, represent a prototype of host-evasion systems found in several pathogenic parasites. However, our knowledge of how antigenic variation operates in trypanosomes remains limited in three vital areas: 1) how expression of a single active VSG is maintained, 2) how the remainder of thousands of VSGs remain silenced, and 3) how their expression status switches. The methodology developed in this proposal by integrating basic genetic concepts and state-of-the-art techniques will efficiently identify genes that regulate specific aspects of monoallelic VSG expression and VSG switching. I will utilize a dual-color reporter strain containing a GFP gene (green) integrated at the transcriptionally active VSG locus and a tdTomato (red) at a silent VSG locus. Two genome-wide libraries, overexpression ORF library containing ~ 7,500 genes (Aim 1) and RNAi library (Aim 2), will be screened for differential expression of GFP and/or tdTomato reporters. If a gene is a critical factor for monoallelic VSG expression and/or VSG switching, depletion or overexpression of this gene should change the expression of reporters. To maximize chances of obtaining true positives, tailored approaches will be used in ORF and RNAi library screens. The overexpression ORF library consists of 30 mini libraries, each containing about 150-350 independent ORFs. Due to this low complexity, ORF library can be screened in a traditional way. ORF library transfected cells will be distributed in 96-well plates. Upon inducing overexpression, each well will be analyzed by flow cytometry and positive clones will be identified by PCR and sequencing. Because RNAi library represents the genome of T. brucei with about 100,000 clones (too many to be screened by manual plating), positive clones will be enriched using two different methods: (i) enrichment of candidate populations exhibiting altered reporter expression by FACS sorting and (ii) enrichment of candidate population by depleting ?non-candidate (parental)? population by Magnet-Activated Cell Sorting (MACS) technique. High-throughput sequencing will clone the genes responsible for differential expression of the reporters from these sorted or depleted populations. Using these integrative approaches, I will identify genes necessary for antigenic variation of T. brucei. Knowledge obtained from this proposal will help uncover molecular mechanisms of allelic exclusion and VSG switching, and also help identify novel drugs that target antigenic variation to prevent disease development.